Microtubule dynamics during cell polarity and migration

描述（由申请人提供）：细胞骨架的极性对许多细胞行为至关重要，包括伤口愈合和趋化过程中的定向迁移。微管聚合动力学在平面极化、迁移的细胞中受时空调控，并且是细胞定向迁移所必需的。本提案的总体目标是确定微管在上皮细胞平面极性和定向片迁移的建立和维持过程中的作用机制，重点研究+TIPs的CLASP家族的功能和动力学。+TIPs是一组异质性蛋白，由它们在细胞中生长的微管+端动态定位而定义。+TIPs是介导上皮片迁移过程中微管功能的主要候选者，因为它们可能为微管末端与皮质细胞骨架的调节相互作用提供界面。目前的研究重点是CLASPs，因为它们与其他+TIPs不同，它们与迁移上皮细胞中微管的时空调节相关。虽然CLASPs在细胞体中跟踪微管+端，但与其他+TIPs相反，CLASPs在迁移上皮细胞的片层中沿着微管结合。通过糖原合成酶激酶3的磷酸化作用，CLASPs沿着片层微管的结合减少了。（GSK3?）是一种新兴的细胞极性通路整合体。因为GSK3 ?被认为在迁移细胞的前部局部失活，预测细胞体中clasp -磷酸化降低了clasp -微管的亲和力，而非磷酸化的clasp与片层微管的结合稳定了这些微管。该建议的具体假设是，CLASP介导的片层微管稳定以及这些微管与皮质细胞骨架的相互作用对于迁移上皮细胞的持久平面极性是必不可少的。这一假设将通过生物化学方法和先进的活细胞显微镜在克隆人类角质形成细胞中进行检验。在Aim 1中，GSK3？-依赖的CLASP磷酸化将在细胞和体外进行分析，并将测试CLASP与微管或相关蛋白的相互作用是否受到磷酸化的调节。在Aim 2中，clasp介导的微管聚合动力学的时空调节将在平面极化、迁移细胞和体外纯化组分中进行分析。将测试CLASPs是否直接或间接调节微管动力学，以及这是否由GSK3调节？磷酸化。在Aim 3中，通过RNA干扰，CLASP功能将在上皮细胞迁移过程中被抑制，并将通过使用活细胞旋转盘共聚焦显微镜分析片层突出、细胞-基质粘附和极性动力学来确定CLASP如何调节定向细胞迁移。

DESCRIPTION (provided by applicant): Polarity of the cytoskeleton is essential for many cell behaviors, including directed migration during wound healing and chemotaxis. Microtubule polymerization dynamics are spatiotemporally regulated in planar polarized, migrating cells, and are required for cells to migrate directionally. The overall objective of this proposal is to determine the mechanisms by which microtubules function during establishment and maintenance of planar polarity and directed sheet migration of epithelial cells with an emphasis on the function and dynamics of the CLASP family of +TIPs. +TIPs are a heterogeneous group of proteins defined by their dynamic localization to growing microtubule plus ends in cells. +TIPs are prime candidates to mediate microtubule function during epithelial sheet migration because they may provide interfaces for regulated interactions of microtubule ends with the cortical cytoskeleton. The current study focuses on CLASPs because they are distinguished from other +TIPs by their spatiotemporally regulated association with microtubules in migrating epithelial cells. Although CLASPs track microtubule plus ends in the cell body, in contrast to other +TIPs CLASPs associate along microtubules in the lamella of migrating epithelial cells. This association of CLASPs along lamella microtubules is decreased through phosphorylation by glycogen synthase kinase 3? (GSK3?), an emerging integrator of cell polarity pathways. Because GSK3? is thought to be locally inactivated in the front of migrating cells, it is predicted that CLASP-phosphorylation in the cell body decreases CLASP-microtubule affinity, while association of non-phosphorylated CLASPs with lamella microtubules stabilizes these microtubules. The specific hypothesis of this proposal is that CLASP- mediated lamella microtubule stabilization and interactions of these microtubules with the cortical cytoskeleton are essential for persistent planar polarity of migrating epithelial cells. This hypothesis will be tested by biochemical methods and advanced live cell microscopy in clonal human keratinocyte cells. In Aim 1, GSK3?-dependent CLASP phosphorylation will be analyzed in cells and in vitro, and it will be tested whether CLASP interactions with microtubules or associated proteins are regulated by phosphorylation. In Aim 2, CLASP-mediated spatiotemporal regulation of microtubule polymerization dynamics will be analyzed in planar polarized, migrating cells, and in vitro with purified components. It will be tested whether CLASPs directly or indirectly regulate microtubule dynamics and whether this is regulated by GSK3? phosphorylation. In Aim 3, CLASP function will be inhibited in migrating epithelial cells by RNA interference, and it will be determined how CLASPs regulate directed cell migration by analyzing lamella protrusion, cell- matrix adhesion, and polarity dynamics using live cell spinning disk confocal microscopy. PUBLIC HEALTH RELEVANCE: Collective cell migration is essential for many morphogenetic movements and the sheet migration of keratinocytes across the basement membrane after injury. In addition, abnormal regulation of cell migration contributes to metastasis of tumor cells, and collective migration of tumor cells plays a crucial role in tumor invasiveness. Understanding the molecular mechanisms underlying planar epithelial cell polarity and directed migration is thus crucial to developing improved therapies for keratinocyte or other carcinomas, and for skin regeneration after wounding.